Collagen producing plants and methods of generating and using same

ABSTRACT

A method of producing collagen in a plant and plants producing collagen are provided. The method is effected by expressing in the plant at least one type of a collagen alpha chain in a manner enabling accumulation of the collagen alpha chain in a subcellular compartment devoid of endogenous P4H activity, thereby producing the collagen in the plant.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/730,071 filed on Mar. 29, 2007, which is a continuation-in-part ofPCT Patent Application No. PCT/IL2005/001045 filed on Sep. 28, 2005,which claims the benefit of priority of U.S. Provisional PatentApplication No. 60/613,719 filed on Sep. 29, 2004. The contents of allof the above applications are incorporated by reference as if fully setforth herein.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to collagen producing plants and methodsof generating and using same. More particularly, the present inventionrelates to a novel approach for generating plants capable of producinghigh levels of hydroxylated collagen chains which are capable of formingnative triple helix type I collagen fibers.

Collagens are the main structural proteins responsible for thestructural integrity of vertebrates and many other multicellularorganisms. Type I collagen represents the prototypical fibrillarcollagen and is the major collagen type in most tissues.

Type I collagen is the predominant collagen component of bone and tendonand is found in large amounts in skin, aorta, and lung. Type I collagenfibers provide great tensile strength and limited extensibility. Themost abundant molecular form of type I collagen is a heterotrimercomposed of two different alpha chains [alpha 1(I)]₂ and alpha 2(I)(Inkinen, 2003). All fibrillar collagen molecules contain threepolypeptide chains constructed from a repeating Gly-X-Y triplet, where Xand Y can be any amino acid but are frequently the imino acids prolineand hydroxyproline.

Fibril forming collagens are synthesized as precursor procollagenscontaining globular N- and C-terminal extension propeptides. Thebiosynthesis of procollagen is a complex process involving a number ofdifferent post-translational modifications including proline and lysinehydroxylation, N-linked and O-linked glycosylation and both intra- andinter-chain disulphide-bond formation. The enzymes carrying out thesemodifications act in a coordinated fashion to ensure the folding andassembly of a correctly aligned and thermally stable triple-helicalmolecule.

Each procollagen molecule assembles within the rough endoplasmicreticulum from the three constituent polypeptide chains. As thepolypeptide chain is co-translationally translocated across the membraneof the endoplasmic reticulum, hydroxylation of proline and lysineresidues occurs within the Gly-X-Y repeat region. Once the polypeptidechain is fully translocated into the lumen of the endoplasmic reticulumthe C-propeptide folds. Three pro-alpha chains then associate via theirC-propeptides to form a trimeric molecule allowing the Gly-X-Y repeatregion to form a nucleation point at its C-terminal end, ensuringcorrect alignment of the chains. The Gly-X-Y region then folds in aC-to-N direction to form a triple helix.

The temporal relationship between polypeptide chain modification andtriple-helix formation is crucial as hydroxylation of proline residuesis required to ensure stability of the triple helix at body temperature,once formed, the triple helix no longer serves as a substrate for thehydroxylation enzyme. The C-propeptides (and to a lesser extent theN-propeptides) keep the procollagen soluble during its passage throughthe cell (Bulleid et al., 2000). Following or during secretion ofprocollagen molecules into the extracellular matrix, propeptides areremoved by procollagen N- and C-proteinases, thereby triggeringspontaneous self-assembly of collagen molecules into fibrils (Hulmes,2002). Removal of the propeptides by procollagen N- and C-proteinaseslowers the solubility of procollagen by >10000-fold and is necessary andsufficient to initiate the self-assembly of collagen into fibers.Crucial to this assembly process are short non triple-helical peptidescalled telopeptides at the ends of the triple-helical domain, whichensure correct registration of the collagen molecules within the fibrilstructure and lower the critical concentration for self-assembly(Bulleid et al., 2000). In nature, the stability of the triple-helicalstructure of collagen requires the hydroxylation of prolines by theenzyme prolyl-4-hydroxylase (P4H) to form residues of hydroxyprolinewithin a collagen chain.

Plants expressing collagen chains are known in the art, see for example,U.S. Pat. No. 6,617,431 and (Merle et al., 2002, Ruggiero et al., 2000).Although plants are capable of synthesizing hydroxyproline-containingproteins the prolyl hydroxylase that is responsible for synthesis ofhydroxyproline in plant cells exhibits relatively loose substratesequence specificity as compared with mammalian P4H and thus, productionof collagen containing hydroxyproline only in the Y position of Gly-X-Ytriplets requires plant co-expression of collagen and P4H genes (Olsenet al, 2003).

An attempt to produce human collagens that rely on the hydroxylationmachinery naturally present in plants resulted in collagen that is poorin proline hydroxylation (Merle et al., 2002). Such collagen melts orloses its triple helical structure at temperatures below 30° C.Co-expression of collagen and prolyl- hydroxylase results with stablehydroxylated collagen that is biologically relevant for applications atbody temperatures (Merle et al., 2002).

Lysyl hydroxylase (LH,EC 1.14.11.4), galactosyltransferase (EC 2.4.1.50)and glucosyltransferase (EC 2.4.1.66) are enzymes involved inposttranslational modifications of collagens. They sequentially modifylysyl residues in specific positions to hydroxylysyl,galactosylhydroxylysyl and glucosylgalactosyl hydroxylysyl residues.These structures are unique to collagens and essential for theirfunctional activity (Wang et al, 2002). A single human enzyme, Lysylhydroxylase 3 (LH3) can catalyze all three consecutive steps inhydroxylysine linked carbohydrate formation (Wang et al, 2002).

Hydroxylysins of a human collagen expressed in tobacco form less than 2%of the hydroxylysins found in a bovine collagen (0.04% of residues/1.88%of residues). This suggests that plant endogenic Lysyl hydroxylase isunable to sufficiently hydroxylate lysines in collagen.

While reducing the present invention to practice, the present inventorsuncovered that efficient hydroxylation of collagen chains relies uponsequestering of the collagen chain along with an enzyme capable ofcorrectly modifying this polypeptide.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided amethod of producing collagen in a plant or an isolated plant cellcomprising expressing in the plant or the isolated plant cell at leastone type of a collagen alpha chain and exogenous P4H in a mannerenabling accumulation of the at least one type of the collagen alphachain and the exogenous P4H in a subcellular compartment devoid ofendogenous P4H activity, thereby producing the collagen in the plant.According to an additional aspect of the present invention there isprovided

According to further features in preferred embodiments of the inventiondescribed below, the method further comprises expressing exogenous LH3in the subcellular compartment devoid of endogenous P4H activity.

According to still further features in the described preferredembodiments the at least one type of the collagen alpha chain includes asignal peptide for targeting to an apoplast or a vacuole.

According to still further features in the described preferredembodiments the at least one type of the collagen alpha chain is devoidof an ER targeting or retention sequence.

According to still further features in the described preferredembodiments the at least one type of the collagen alpha chain isexpressed in a DNA-containing organelle of the plant.

According to still further features in the described preferredembodiments the exogenous P4H includes a signal peptide for targeting toan apoplast or a vacuole.

According to still further features in the described preferredembodiments the exogenous P4H is devoid of an ER targeting or retentionsequence.

According to still further features in the described preferredembodiments the exogenous P4H is expressed in a DNA-containing organelleof the plant.

According to still further features in the described preferredembodiments the at least one type of the collagen alpha chain is alpha 1chain.

According to still further features in the described preferredembodiments the at least one type of the collagen alpha chain is alpha 2chain.

According to still further features in the described preferredembodiments the at least one type of the collagen alpha chain includes aC-terminus and/or an N-terminus propeptide.

According to still further features in the described preferredembodiments the plant is selected from the group consisting of Tobacco,Maize, Alfalfa, Rice, Potato, Soybean, Tomato, Wheat, Barley, Canola andCotton.

According to still further features in the described preferredembodiments the at least one type of the collagen alpha chain or theexogenous P4H are expressed in only a portion of the plant.

According to still further features in the described preferredembodiments the portion of the plant is leaves, seeds, roots, tubers orstems.

According to still further features in the described preferredembodiments the exogenous P4H is capable of specifically hydroxylatingthe Y position of Gly-X-Y triplets of the at least one type of thecollagen alpha chain.

According to still further features in the described preferredembodiments the exogenous P4H is human P4H.

According to still further features in the described preferredembodiments the plant is subjected to a stress condition.

According to still further features in the described preferredembodiments the stress condition is selected from the group consistingof drought, salinity, injury, cold and spraying with stress inducingcompounds.

According to another aspect of the present invention there is provided agenetically modified plant or isolated plant cell capable ofaccumulating a collagen alpha chain having a hydroxylation patternidentical to that produced when the collagen alpha chain is expressed inhuman cells.

According to yet another aspect of the present invention there isprovided a genetically modified plant or isolated plant cell capable ofaccumulating a collagen alpha chain in a subcellular compartment devoidof endogenous P4H activity.

According to still further features in the described preferredembodiments the genetically modified plant further comprises anexogenous P4H.

According to still further features in the described preferredembodiments the at least one type of the collagen alpha chain includes asignal peptide for targeting to an apoplast or a vacuole.

According to still further features in the described preferredembodiments the at least one type of the collagen alpha chain is devoidof an ER targeting or retention sequence.

According to still further features in the described preferredembodiments the at least one type of the collagen alpha chain isexpressed in a DNA-containing organelle of the plant.

According to still further features in the described preferredembodiments the exogenous P4H includes a signal peptide for targeting toan apoplast or a vacuole.

According to still further features in the described preferredembodiments the exogenous P4H is devoid of an ER targeting or retentionsequence.

According to still further features in the described preferredembodiments the exogenous P4H is expressed in a DNA-containing organelleof the plant.

According to still further features in the described preferredembodiments the collagen alpha chain is alpha 1 chain.

According to still further features in the described preferredembodiments the collagen alpha chain is alpha 2 chain.

According to still further features in the described preferredembodiments the collagen alpha chain includes a C-terminus and/or anN-terminus propeptide.

According to still another aspect of the present invention there isprovided a plant system comprising a first genetically modified plantcapable of accumulating a collagen alpha 1 chain and a secondgenetically modified plant capable of accumulating a collagen alpha 2chain.

According to yet another aspect of the present invention there isprovided a plant system comprising a first genetically modified plantcapable of accumulating a collagen alpha 1 chain and a collagen alpha 2chain and a second genetically modified plant capable of accumulatingP4H.

According to still further features in the described preferredembodiments at least one of the first genetically modified plant and thesecond genetically modified plant further comprises exogenous P4H.

According to yet another aspect of the present invention there isprovided a method of producing fibrillar collagen comprising: (a)expressing in a first plant a collagen alpha 1 chain; (b) expressing ina second plant a collagen alpha 2 chain, wherein expression in the firstplant and the second plant the is configured such that the collagenalpha 1 chain and the collagen alpha 2 chain are each capable ofaccumulating in a subcellular compartment devoid of endogenous P4Hactivity; and (c) crossing the first plant and the second plant andselecting progeny expressing the collagen alpha 1 chain and the collagenalpha 2 chain thereby producing fibrillar collagen.

According to still further features in the described preferredembodiments the method further comprises expressing an exogenous P4H ineach of the first plant and the second plant.

According to still further features in the described preferredembodiments each of the collagen alpha 1 chain and the collagen alpha 2chain includes a signal peptide for targeting to an apoplast or avacuole.

According to still further features in the described preferredembodiments each of the collagen alpha 1 chain and the collagen alpha 2chain is devoid of an ER targeting or retention sequence.

According to still further features in the described preferredembodiments steps (a) and (b) are effected via expression in aDNA-containing organelle of the plant.

According to still further features in the described preferredembodiments the exogenous P4H includes a signal peptide for targeting toan apoplast or a vacuole.

According to still further features in the described preferredembodiments the exogenous P4H is devoid of an ER targeting or retentionsequence.

According to still further features in the described preferredembodiments the exogenous P4H is expressed in a DNA-containing organelleof the plant.

According to still further features in the described preferredembodiments each of the collagen alpha 1 chain and the collagen alpha 2chain includes a C-terminus and/or an N-terminus propeptide.

According to still further features in the described preferredembodiments the exogenous P4H is capable of specifically hydroxylatingthe Y position of Gly-X-Y triplets of the at least one type of thecollagen alpha chain.

According to still further features in the described preferredembodiments the exogenous P4H is human P4H.

According to still further features in the described preferredembodiments the first plant and the second plant are subjected to astress condition.

According to still further features in the described preferredembodiments the stress condition is selected from the group consistingof drought, salinity, injury, heavy metal toxicity and cold stress.

According to yet another aspect of the present invention there isprovided a method of producing fibrillar collagen comprising: (a)expressing in a first plant a collagen alpha 1 chain and a collagenalpha 2 chain, wherein expression in the first plant is configured suchthat the collagen alpha 1 chain and the collagen alpha 2 chain are eachcapable of accumulating in a subcellular compartment devoid ofendogenous P4H activity; (b) expressing in a second plant an exogenousP4H capable of accumulating in the subcellular compartment devoid ofendogenous P4H activity; and (c) crossing the first plant and the secondplant and selecting progeny expressing the collagen alpha 1 chain, thecollagen alpha 2 chain and the P4H thereby producing fibrillar collagen.

According to yet another aspect of the present invention there isprovided a nucleic acid construct comprising a polynucleotide encoding ahuman P4H positioned under the transcriptional control of a promoterfunctional in plant cells.

According to still further features in the described preferredembodiments the promoter is selected from the group consisting of theCaMV 35S promoter, the Ubiquitin promoter, the rbcS promoter and theSVBV promoter.

According to yet another aspect of the present invention there isprovided a genetically modified plant or isolated plant cell beingcapable of expressing collagen alpha 1 chain, collagen alpha 2 chain,P4H, LH3 and protease C and/or protease N.

According to still further features in the described preferredembodiments the collagen alpha 1 chain and the collagen alpha 2 chainare each capable of accumulating in a subcellular compartment devoid ofendogenous plant P4H activity.

According to yet another aspect of the present invention there isprovided a genetically modified plant or isolated plant cell beingcapable of accumulating collagen having a temperature stabilitycharacteristic identical to that of mammalian collagen.

According to still further features in the described preferredembodiments the collagen is type I collagen.

According to still further features in the described preferredembodiments the mammalian collagen is human collagen.

According to yet another aspect of the present invention there isprovided a collagen-encoding sequence optimized for expression in aplant.

According to still further features in the described preferredembodiments the collagen encoding sequence is as set forth by SEQ IDNO:1.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing a plant capable ofexpressing correctly hydroxylated collagen chains which are capable ofassembling into collagen having properties similar to that of humancollagen.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIGS. 1 a-d illustrate construction of various expression cassettes andvectors used to transform test plants. All of the coding sequencessynthesized as a part of the present study were optimized for expressionin tobacco. FIG. 1 a shows a cloning scheme of type I collagen alpha Ichain or type II collagen alpha 2 chain into a plant expression vectorin accordance with some embodiments of the present invention; FIG. 1 bshows a cloning scheme of the enzyme prolyl-4-hydroxylase (P4H) into aplant expression vector in accordance with some embodiments of thepresent invention; FIG. 1 c shows a cloning scheme proteinase C orproteinase N into a plant expression vector in accordance with someembodiments of the present invention; FIG. 1 d shows a cloning scheme ofLysyl hydroxylase 3 (LH3) into a plant expression vector in accordancewith some embodiments of the present invention. A multiple cloning siteset forth in SEQ ID NO: 29 is shown at the bottom of each panel.

FIG. 2 illustrates various co-transformations approaches. Eachexpression cassette is represented by the short name of the codingsequence. The coding sequences are specified in table 1. Eachco-transformation was performed by two pBINPLUS binary vectors. Eachrectangle represents a single pBINPLUS vector carrying one, two or threeexpression cassettes. Promoter and terminators are specified in Example1.

FIG. 3 is a multiplex PCR screening of transformants showing plants thatare positive for Collagen alpha 1 (324 bp fragment) or Collagen alpha 2(537 bp fragment) or both.

FIG. 4 is western blot analysis of transgenic plants generated byco-transformations 2, 3 and 4. Total soluble proteins were extractedfrom tobacco co-transformants #2, #3 and #4 and tested withanti-Collagen I antibody (#AB745 from Chemicon Inc.). Size markers were#SM0671 from Fermentas Inc. W.T. is a wild type tobacco. Positivecollagen bands are visible in plants that are PCR positive for collagentypeI alpha 1 or alpha 2 or both. Positive control band of 500 ngcollagen type I from human placenta (#CC050 from Chemicon Inc.,extracted from human placenta by pepsin digestion) represents about 0.3%of the total soluble proteins (about 150 μg) in the samples from thetransgenic plants. The larger band at about 140 kDa in the humancollagen sample is a procollagen with it's C-propeptide as detected byanti carboxy-terminal pro-peptide of collagen type I antibody (#MAB 1913from Chemicon Inc.). The smaller band at about 120 kDa in the humancollagen sample is a collagen without propeptides. Due to their unusualcomposition proline rich proteins (including collagen)s consistentlymigrate on polyacrylamid gels as bands with molecular mass higher thanexpected. Therefore the collagen chains without propeptides with amolecular weight of about 95 kDa migrate as a band of about 120 kDa.

FIG. 5 is a western blot analysis of transgenic plant generated byco-transformation #8 (carrying appoplast signals translationally fusedto the collagen chains). Total soluble proteins were extracted fromtransgenic tobacco leaves and tested with anti-Collagen I antibody(#AB745 from Chemicon Inc.) Positive collagen alpha 2 band is visible inplant 8-141. Collagen type I from human placenta (#CC050 from ChemiconInc.) served as control.

FIGS. 6 a-b illustrate collagen triple helix assembly and thermalstability as qualified by heat treatment and Trypsin or Pepsindigestion. In FIG. 6 a—total soluble protein from tobacco 2-9(expressing only col alpha1 and no P4H) and 3-5 (expressing both colalpha 1+2 and human P4H alpha and beta subunits) were subjected to heattreatment (15 minutes in 38° C. or 43° C.) followed by Trypsin digestion(20 minutes in R.T.) and tested with anti-Collagen I antibody in aWestern blot procedure. Positive controls were samples of 500 ng humancollagen I+total soluble proteins of w.t. tobacco. In FIG. 6 b—totalsoluble proteins were extracted from transgenic tobacco 13-6 (expressingcollagen I alpha 1 and alpha 2 chains—pointed by arrows, human P4H alphaand beta subunits and human LH3) and subjected to heat treatment (20minutes in 33° C., 38° C. or 42° C.), immediately cooled on ice toprevent reassembly of triple helix and incubated with pepsin for 30minutes in room temperature (about 22° C.) followed by testing withanti-Collagen I antibody ((#AB745 from Chemicon Inc.) in a standardWestern blot procedure. Positive control was sample of ˜50 ng humancollagen I (#CC050 from Chemicon Inc., extracted from human placenta bypepsin digestion) which was added to total soluble proteins extractedfrom w.t. tobacco.

FIG. 7 illustrates Northern blot analysis conducted on wild typetobacco. Blots were probed with tobacco P4H cDNA.

FIG. 8 is a western blot analysis of transgenic plants generated byco-transformations 2, 3 and 13. Total soluble protein was extracted fromtobacco co-transformants and tested with anti human P4H alpha and betaand anti-Collagen I antibodies.

FIG. 9 is a western blot analysis of (lane 1) cross breeding vacuolartargeted plants A(2-300♀+20-279♂) grown under normal light regimen; and13-652 vacuolar targeted plants grown for 8 days in the dark. All plantsexpress exogenous col1, col2, P4H α and β as well as LH3 (PCRvalidated).

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention is of plants expressing and accumulating collagenwhich can be used to produce collagen and collagen fibers which displaycharacteristics of mammalian collagen.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

Collagen producing plants are known in the art. Although such plants canbe used to produce collagen chains as well as collagen, such chains areincorrectly hydroxylated and thus self-assembly thereof, whether inplanta or not, leads to collagen which is inherently unstable.

While reducing the present invention to practice, the present inventorshave devised a plant expression approach which ensures correcthydroxylation of collagen chains and thus enables in-planta productionof collagen which closely mimics the characteristics (e.g. temperaturestability) of human type I collagen.

Thus, according to one aspect of the present invention there is provideda genetically modified plant which is capable of expressing at least onetype of a collagen alpha chain and accumulating it in a subcellularcompartment which is devoid of endogenous P4H activity.

As used herein, the phrase “genetically modified plant” refers to anylower (e.g. moss) or higher (vascular) plant or a tissue or an isolatedcell thereof (e.g., of a cell suspension) which is stably or transientlytransformed with an exogenous polynucleotide sequence. Examples ofplants include Tobacco, Maize, Alfalfa, Rice, Potato, Soybean, Tomato,Wheat, Barley, Canola, Cotton, Carrot as well as lower plants such asmoss.

As used herein, the phrase “collagen chain” refers to a collagen subunitsuch as the alpha 1 or 2 chains of collagen fibers, preferably type Ifibers. As used herein, the phrase “collagen ” refers to an assembledcollagen trimer, which in the case of type I collagen includes two alpha1 chains and one alpha 2 chain. A collagen fiber is collagen which isdevoid of terminal propeptides C and N.

As is used herein, the phrase “subcellular compartment devoid ofendogenous P4H activity” refers to any compartmentalized region of thecell which does not include plant P4H or an enzyme having plant-like P4Hactivity. Examples of such subcellular compartments include the vacuole,apoplast and cytoplasm as well as organelles such as the chloroplast,mitochondria and the like.

Any type of collagen chain can be expressed by the genetically modifiedplant of the present invention. Examples include Fibril-formingcollagens (types I, II, III, V, and XI), networks forming collagens(types IV, VIII, and X), collagens associated with fibril surfaces(types IX, XII, and XIV), collagens which occur as transmembraneproteins (types XIII and XVII), or form 11-nm periodic beaded filaments(type VI). For further description please see Hulmes, 2002.

Preferably, the collagen chain expressed is an alpha 1 and/or 2 chain oftype I collagen. The expressed collagen alpha chain can be encoded byany polynucleotide sequences derived from any mammal. Preferably, thesequences encoding collagen alpha chains are human and are set forth bySEQ ID NOs: 1 and 4.

Typically, alpha collagen chains expressed in plants may or may notinclude their terminal propeptides (i.e. propeptide C and propeptide N).

Ruggiero et al. (2000) note that processing of procollagen by plantproteolytic activity is different then normal processing in human andthat propeptide C is removed by plant proteolytic activity although thecleavage site is unknown. Cleavage of the C propeptide may take place ona procollagen peptide before the assembly of trimmer (association ofthree C-Propeptides is essential for initiating the assembly oftrimmers).

N-propeptide cleavage by plant proteolytic activity takes place inmature plants but not in plantlets. Such cleavage removes 2 amino acidsfrom the N telopeptide (2 out of 17).

The C-propeptides (and to a lesser extent the N-propeptides) maintainthe procollagen soluble during its passage through the animal cell(Bulleid et al., 2000) and are expected to have a similar effect in theplant cell. Following or during secretion of procollagen molecules intothe extracellular matrix, propeptides are removed by procollagen N- andC-proteinases, thereby triggering spontaneous self-assembly of collagenmolecules into fibrils (Hulmes, 2002). Removal of the propeptides byprocollagen N- and C-proteinases lowers the solubility of procollagenby >10000-fold and is necessary and sufficient to initiate theself-assembly of collagen into fibers. Crucial to this assembly processare short non triple-helical peptides called telopeptides at the ends ofthe triple-helical domain, which ensure correct registration of thecollagen molecules within the fibril structure and lower the criticalconcentration for self-assembly (Bulleid et al., 2000). Prior artdescribe the use of pepsin to cleave the propeptides during productionof collagen (Bulleid et al 2000). However pepsin damages thetelopeptides and as a result, pepsin-extracted collagen is unable toform ordered fibrillar structures (Bulleid et al 2000).

Protein disulfide isomerase (PDI) that form the beta subunit of humanP4H was shown to bind to the C-propeptide prior to trimmer assemblythereby also acting as a molecular chaperone during chain assembly(Ruggiero et al, 2000).

The use of human Procollagen I N-proteinase and Procollagen C-proteinaseexpressed in a different plants may generate collagen that is moresimilar to the native human collagen and can form ordered fibrillarstructures.

In a case where N or C propeptides or both are included in the expressedcollagen chain, the genetically modified plant of the present inventioncan also express the respective protease (i.e. C or N or both).Polynucleotide sequences encoding such proteases are exemplified by SEQID NOs: 18 (protease C) and 20 (Protease N). Such proteases can beexpressed such that they are accumulated in the same subcellularcompartment as the collagen chain.

Accumulation of the expressed collagen chain in a subcellularcompartment devoid of endogenous P4H activity can be effected via anyone of several approaches.

For example, the expressed collagen chain can include a signal sequencefor targeting the expressed protein to a subcellular compartment such asthe apoplast or an organelle (e.g. chloroplast). Examples of suitablesignal sequences include the chloroplast transit peptide (included inSwiss-Prot entry P07689, amino acids 1-57) and the Mitochondrion transitpeptide (included in Swiss-Prot entry P46643, amino acids 1-28). TheExamples section which follows provides additional examples of suitablesignal sequences as well as guidelines for employing such signalsequences in expression of collagen chains in plant cells.

Alternatively, the sequence of the collagen chain can be modified in away which alters the cellular localization of collagen when expressed inplants.

As is mentioned hereinabove, the ER of plants includes a P4H which isincapable of correctly hydroxylating collagen chains. Collagen alphachains natively include an ER targeting sequence which directs expressedcollagen into the ER where it is post-translationally modified(including incorrect hydroxylation). Thus, removal of the ER targetingsequence will lead to cytoplasmic accumulation of collagen chains whichare devoid of post translational modification including anyhydroxylations.

Example 1 of the Examples section which follows describes generation ofcollagen sequences which are devoid of ER sequences.

Still alternatively, collagen chains can be expressed and accumulated ina DNA containing organelle such as the chloroplast or mitochondria.Further description of chloroplast expression is provided hereinbelow.

As is mentioned hereinabove, hydroxylation of alpha chains is requiredfor assembly of a stable type I collagen. Since alpha chains expressedby the genetically modified plant of the present invention accumulate ina compartment devoid of endogenous P4H activity, such chains must beisolated from the plant, plant tissue or cell and in-vitro hydroxylated.Such hydroxylation can be achieved by the method described byTurpeenniemi-Hujanen and Myllyla (Concomitant hydroxylation of prolineand lysine residues in collagen using purified enzymes in vitro. BiochimBiophys Acta. 1984 Jul. 16; 800(1):59-65).

Although such in-vitro hydroxylation can lead to correctly hydroxylatedcollagen chains, it can be difficult and costly to achieve.

To overcome the limitations of in-vitro hydroxylation, the geneticallymodified plant of the present invention preferably also co-expresses P4Hwhich is capable of correctly hydroxylating the collagen alpha chain(s)[i.e. hydroxylating only the proline (Y) position of the Gly-X-Ytriplets]. P4H is an enzyme composed of two subunits, alpha and beta.Both are needed to form an active enzyme while the Beta subunit alsoposses a chaperon function.

The P4H expressed by the genetically modified plant of the presentinvention is preferably a human P4H which is encoded by, for example,SEQ ID's NO:12 and 14. In addition, P4H mutants which exhibit enhancedsubstrate specificity, or P4H homologues can also be used.

A suitable P4H homologue is exemplified by an Arabidopsis oxidoreductaseidentified by NCBI accession NP_(—)179363. Pairwise alignment of thisprotein sequence and a human P4H alpha subunit conducted by the presentinventors revealed the highest homology between functional domains ofany known P4H homologs of plants.

Since P4H needs to co-accumulate with the expressed collagen chain, thecoding sequence thereof is preferably modified accordingly (addition ofsignal sequences, deletions which may prevent ER targeting etc).

In mammalian cells, collagen is also modified by Lysyl hydroxylase,galactosyltransferase and glucosyltransferase. These enzymessequentially modify lysyl residues in specific positions tohydroxylysyl, galactosylhydroxylysyl and glucosylgalactosyl hydroxylysylresidues. A single human enzyme, Lysyl hydroxylase 3 (LH3) can catalyzeall three consecutive steps in hydroxylysine linked carbohydrateformation.

Thus, the genetically modified plant of the present invention preferablyalso expresses mammalian LH3. An LH3 encoding sequence such as that setforth by SEQ ID NO: 22 can be used for such purposes.

The collagen chain(s) and modifying enzymes described above can beexpressed from a stably integrated or a transiently expressed nucleicacid construct which includes polynucleotide sequences encoding thealpha chains and/or modifying enzymes (e.g. P4H and LH3) positionedunder the transcriptional control of plant functional promoters. Such anucleic acid construct (which is also termed herein as an expressionconstruct) can be configured for expression throughout the whole plant,defined plant tissues or defined plant cells, or at define developmentalstages of the plant. Such a construct may also include selection markers(e.g. antibiotic resistance), enhancer elements and an origin ofreplication for bacterial replication.

It will be appreciated that constructs including two expressible inserts(e.g. two alpha chain types, or an alpha chain and P4H) preferablyinclude an individual promoter for each insert, or alternatively suchconstructs can express a single transcript chimera including both insertsequences from a single promoter. In such a case, the chimerictranscript includes an IRES sequence between the two insert sequencessuch that the downstream insert can be translated therefrom.

Numerous plant functional expression promoters and enhancers which canbe either tissue specific, developmentally specific, constitutive orinducible can be utilized by the constructs of the present invention,some examples are provided hereinunder.

As used herein in the specification and in the claims section thatfollows the phrase “plant promoter” or “promoter” includes a promoterwhich can direct gene expression in plant cells (including DNAcontaining organelles). Such a promoter can be derived from a plant,bacterial, viral, fungal or animal origin. Such a promoter can beconstitutive, i.e., capable of directing high level of gene expressionin a plurality of plant tissues, tissue specific, i.e., capable ofdirecting gene expression in a particular plant tissue or tissues,inducible, i.e., capable of directing gene expression under a stimulus,or chimeric, i.e., formed of portions of at least two differentpromoters.

Thus, the plant promoter employed can be a constitutive promoter, atissue specific promoter, an inducible promoter or a chimeric promoter.

Examples of constitutive plant promoters include, without being limitedto, CaMV35S and CaMV19S promoters, FMV34S promoter, sugarcanebacilliform badnavirus promoter, CsVMV promoter, Arabidopsis ACT2/ACT8actin promoter, Arabidopsis ubiquitin UBQ1 promoter, barley leaf thioninBTH6 promoter, and rice actin promoter.

Examples of tissue specific promoters include, without being limited to,bean phaseolin storage protein promoter, DLEC promoter, PHS promoter,zein storage protein promoter, conglutin gamma promoter from soybean,AT2S 1 gene promoter, ACT11 actin promoter from Arabidopsis, napApromoter from Brassica napus and potato patatin gene promoter.

The inducible promoter is a promoter induced by a specific stimuli suchas stress conditions comprising, for example, light, temperature,chemicals, drought, high salinity, osmotic shock, oxidant conditions orin case of pathogenicity and include, without being limited to, thelight-inducible promoter derived from the pea rbcS gene, the promoterfrom the alfalfa rbcS gene, the promoters DRE, MYC and MYB active indrought; the promoters INT, INPS, prxEa, Ha hsp17.7G4 and RD21 active inhigh salinity and osmotic stress, and the promoters hsr203J and str246Cactive in pathogenic stress.

Preferably the promoter utilized by the present invention is a strongconstitutive promoter such that over expression of the construct insertsis effected following plant transformation.

It will be appreciated that any of the construct types used in thepresent invention can be co-transformed into the same plant using sameor different selection markers in each construct type. Alternatively thefirst construct type can be introduced into a first plant while thesecond construct type can be introduced into a second isogenic plant,following which the transgenic plants resultant therefrom can be crossedand the progeny selected for double transformants. Further self-crossesof such progeny can be employed to generate lines homozygous for bothconstructs.

There are various methods of introducing nucleic acid constructs intoboth monocotyledonous and dicotyledenous plants (Potrykus, I., Annu.Rev. Plant. Physiol., Plant. Mol. Biol. (1991) 42:205-225; Shimamoto etal., Nature (1989) 338:274-276). Such methods rely on either stableintegration of the nucleic acid construct or a portion thereof into thegenome of the plant, or on transient expression of the nucleic acidconstruct in which case these sequences are not inherited by a progenyof the plant.

In addition, several method exist in which a nucleic acid construct canbe directly introduced into the DNA of a DNA containing organelle suchas a chloroplast.

There are two principle methods of effecting stable genomic integrationof exogenous sequences such as those included within the nucleic acidconstructs of the present invention into plant genomes:

(i) Agrobacterium-mediated gene transfer: Klee et al. (1987) Annu. Rev.Plant Physiol. 38:467-486; Klee and Rogers in Cell Culture and SomaticCell Genetics of Plants, Vol. 6, Molecular Biology of Plant NuclearGenes, eds. Schell, J., and Vasil, L. K., Academic Publishers, SanDiego, Calif. (1989) p. 2-25; Gatenby, in Plant Biotechnology, eds.Kung, S. and Arntzen, C. J., Butterworth Publishers, Boston, Mass.(1989) p. 93-112.

(ii) direct DNA uptake: Paszkowski et al., in Cell Culture and SomaticCell Genetics of Plants, Vol. 6, Molecular Biology of Plant NuclearGenes eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego,Calif. (1989) p. 52-68; including methods for direct uptake of DNA intoprotoplasts, Toriyama, K. et al. (1988) Bio/Technology 6:1072-1074. DNAuptake induced by brief electric shock of plant cells: Zhang et al.Plant Cell Rep. (1988) 7:379-384. Fromm et al. Nature (1986)319:791-793. DNA injection into plant cells or tissues by particlebombardment, Klein et al. Bio/Technology (1988) 6:559-563; McCabe et al.Bio/Technology (1988) 6:923-926; Sanford, Physiol. Plant. (1990)79:206-209; by the use of micropipette systems: Neuhaus et al., Theor.Appl. Genet. (1987) 75:30-36; Neuhaus and Spangenberg, Physiol. Plant.(1990) 79:213-217; or by the direct incubation of DNA with germinatingpollen, DeWet et al. in Experimental Manipulation of Ovule Tissue, eds.Chapman, G. P. and Mantell, S. H. and Daniels, W. Longman, London,(1985) p. 197-209; and Ohta, Proc. Natl. Acad. Sci. USA (1986)83:715-719.

The Agrobacterium system includes the use of plasmid vectors thatcontain defined DNA segments that integrate into the plant genomic DNA.Methods of inoculation of the plant tissue vary depending upon the plantspecies and the Agrobacterium delivery system. A widely used approach isthe leaf disc procedure which can be performed with any tissue explantthat provides a good source for initiation of whole plantdifferentiation. Horsch et al. in Plant Molecular Biology Manual A5,Kluwer Academic Publishers, Dordrecht (1988) p. 1-9. A supplementaryapproach employs the Agrobacterium delivery system in combination withvacuum infiltration. The Agrobacterium system is especially viable inthe creation of transgenic dicotyledenous plants.

There are various methods of direct DNA transfer into plant cells. Inelectroporation, protoplasts are briefly exposed to a strong electricfield. In microinjection, the DNA is mechanically injected directly intothe cells using very small micropipettes. In microparticle bombardment,the DNA is adsorbed on microprojectiles such as magnesium sulfatecrystals, tungsten particles or gold particles, and the microprojectilesare physically accelerated into cells or plant tissues.

Following transformation plant propagation is exercised. The most commonmethod of plant propagation is by seed. Regeneration by seedpropagation, however, has the deficiency that due to heterozygositythere is a lack of uniformity in the crop, since seeds are produced byplants according to the genetic variances governed by Mendelian rules.Basically, each seed is genetically different and each will grow withits own specific traits. Therefore, it is preferred that the transformedplant be produced such that the regenerated plant has the identicaltraits and characteristics of the parent transgenic plant. Therefore, itis preferred that the transformed plant be regenerated bymicropropagation which provides a rapid, consistent reproduction of thetransformed plants.

Transient expression methods which can be utilized for transientlyexpressing the isolated nucleic acid included within the nucleic acidconstruct of the present invention include, but are not limited to,microinjection and bombardment as described above but under conditionswhich favor transient expression, and viral mediated expression whereina packaged or unpackaged recombinant virus vector including the nucleicacid construct is utilized to infect plant tissues or cells such that apropagating recombinant virus established therein expresses thenon-viral nucleic acid sequence.

Viruses that have been shown to be useful for the transformation ofplant hosts include CaMV, TMV and BV. Transformation of plants usingplant viruses is described in U.S. Pat. No. 4,855,237 (BGV), EP-A 67,553(TMV), Japanese Published Application No. 63-14693 (TMV), EPA 194,809(BV), EPA 278,667 (BV); and Gluzman, Y. et al., Communications inMolecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, NewYork, pp. 172-189 (1988). Pseudovirus particles for use in expressingforeign DNA in many hosts, including plants, is described in WO87/06261.

Construction of plant RNA viruses for the introduction and expression ofnon-viral exogenous nucleic acid sequences in plants is demonstrated bythe above references as well as by Dawson, W. O. et al., Virology (1989)172:285-292; Takamatsu et al. EMBO J. (1987) 6:307-311; French et al.Science (1986) 231:1294-1297; and Takamatsu et al. FEBS Letters (1990)269:73-76.

When the virus is a DNA virus, the constructions can be made to thevirus itself. Alternatively, the virus can first be cloned into abacterial plasmid for ease of constructing the desired viral vector withthe foreign DNA. The virus can then be excised from the plasmid. If thevirus is a DNA virus, a bacterial origin of replication can be attachedto the viral DNA, which is then replicated by the bacteria.Transcription and translation of this DNA will produce the coat proteinwhich will encapsidate the viral DNA. If the virus is an RNA virus, thevirus is generally cloned as a cDNA and inserted into a plasmid. Theplasmid is then used to make all of the constructions. The RNA virus isthen produced by transcribing the viral sequence of the plasmid andtranslation of the viral genes to produce the coat protein(s) whichencapsidate the viral RNA.

Construction of plant RNA viruses for the introduction and expression inplants of non-viral exogenous nucleic acid sequences such as thoseincluded in the construct of the present invention is demonstrated bythe above references as well as in U.S. Pat. No. 5,316,931.

In one embodiment, a plant viral nucleic acid is provided in which thenative coat protein coding sequence has been deleted from a viralnucleic acid, a non-native plant viral coat protein coding sequence anda non-native promoter, preferably the subgenomic promoter of thenon-native coat protein coding sequence, capable of expression in theplant host, packaging of the recombinant plant viral nucleic acid, andensuring a systemic infection of the host by the recombinant plant viralnucleic acid, has been inserted. Alternatively, the coat protein genemay be inactivated by insertion of the non-native nucleic acid sequencewithin it, such that a protein is produced. The recombinant plant viralnucleic acid may contain one or more additional non-native subgenomicpromoters. Each non-native subgenomic promoter is capable oftranscribing or expressing adjacent genes or nucleic acid sequences inthe plant host and incapable of recombination with each other and withnative subgenomic promoters. Non-native (foreign) nucleic acid sequencesmay be inserted adjacent the native plant viral subgenomic promoter orthe native and a non-native plant viral subgenomic promoters if morethan one nucleic acid sequence is included. The non-native nucleic acidsequences are transcribed or expressed in the host plant under controlof the subgenomic promoter to produce the desired products.

In a second embodiment, a recombinant plant viral nucleic acid isprovided as in the first embodiment except that the native coat proteincoding sequence is placed adjacent one of the non-native coat proteinsubgenomic promoters instead of a non-native coat protein codingsequence.

In a third embodiment, a recombinant plant viral nucleic acid isprovided in which the native coat protein gene is adjacent itssubgenomic promoter and one or more non-native subgenomic promoters havebeen inserted into the viral nucleic acid. The inserted non-nativesubgenomic promoters are capable of transcribing or expressing adjacentgenes in a plant host and are incapable of recombination with each otherand with native subgenomic promoters. Non-native nucleic acid sequencesmay be inserted adjacent the non-native subgenomic plant viral promoterssuch that said sequences are transcribed or expressed in the host plantunder control of the subgenomic promoters to produce the desiredproduct.

In a fourth embodiment, a recombinant plant viral nucleic acid isprovided as in the third embodiment except that the native coat proteincoding sequence is replaced by a non-native coat protein codingsequence.

The viral vectors are encapsidated by the coat proteins encoded by therecombinant plant viral nucleic acid to produce a recombinant plantvirus. The recombinant plant viral nucleic acid or recombinant plantvirus is used to infect appropriate host plants. The recombinant plantviral nucleic acid is capable of replication in the host, systemicspread in the host, and transcription or expression of foreign gene(s)(isolated nucleic acid) in the host to produce the desired protein.

A technique for introducing exogenous nucleic acid sequences to thegenome of the chloroplasts is known. This technique involves thefollowing procedures. First, plant cells are chemically treated so as toreduce the number of chloroplasts per cell to about one. Then, theexogenous nucleic acid is introduced via particle bombardment into thecells with the aim of introducing at least one exogenous nucleic acidmolecule into the chloroplasts. The exogenous nucleic acid is selectedsuch that it is integratable into the chloroplast's genome viahomologous recombination which is readily effected by enzymes inherentto the chloroplast. To this end, the exogenous nucleic acid includes, inaddition to a gene of interest, at least one nucleic acid stretch whichis derived from the chloroplast's genome. In addition, the exogenousnucleic acid includes a selectable marker, which serves by sequentialselection procedures to ascertain that all or substantially all of thecopies of the chloroplast genomes following such selection will includethe exogenous nucleic acid. Further details relating to this techniqueare found in U.S. Pat. Nos. 4,945,050; and 5,693,507 which areincorporated herein by reference. A polypeptide can thus be produced bythe protein expression system of the chloroplast and become integratedinto the chloroplast's inner membrane.

The above described transformation approaches can be used to producecollagen chains and/or modifying enzymes as well as assembled collagen(with or without propeptides) in any species of plant, or plant tissueor isolated plants cell derived therefrom.

Preferred plants are those which are capable of accumulating largeamounts of collagen chains, collagen and/or the processing enzymesdescribed herein. Such plants may also be selected according to theirresistance to stress conditions and the ease at which expressedcomponents or assembled collagen can be extracted. Examples of preferredplants include Tobacco, Maize, Alfalfa, Rice, Potato, Soybean, Tomato,Wheat, Barley, Canola and Cotton.

Collagen fibers are extensively used in the food and cosmetics industry.Thus, although collagen fiber components (alpha chains) and modifyingenzymes expressed by plants find utility in industrial synthesis ofcollagen, complete collagen production in plants is preferred for itssimplicity and cost effectiveness.

Several approaches can be used to generate type I collagen in plants.For example, collagen alpha 1 chain can be isolated from a plantexpressing collagen alpha 1 and P4H (and optionally LH3) and mixed witha collagen alpha 2 chain which is isolated from a plant expressingcollagen alpha 2 and P4H (and optionally LH3 and protease C and/or N).Since collagen alpha 1 chain self assembles into a triple helix byitself, it may be necessary to denature such a homo-trimer prior tomixing and renaturation with the collagen alpha 2 chain.

Preferably, a first plant expressing collagen alpha 1 and P4H (andoptionally LH3 and protease C and/or N) can be crossed with a second(and preferably isogenic) plant which expresses collagen alpha 2 oralternatively, a first plant expressing both alpha chains can be crossedwith a second plant expressing P4H and optionally LH3 and protease Cand/or N.

It should be noted that although the above described plant breedingapproaches utilize two individually transformed plants, approaches whichutilize three or more individually transformed plants, each expressingone or two components can also be utilized.

One of ordinary skill in the art would be well aware of various plantbreeding techniques and as s such no further description of suchtechniques is provided herein.

Although plant breeding approaches are preferred, it should be notedthat a single plant expressing collagen alpha 1 and 2, P4H and LH3 (andoptionally protease C and/or N) can be generated via severaltransformation events each designed for introducing one more expressiblecomponents into the cell. In such cases, stability of eachtransformation event can be verified using specific selection markers.

In any case, transformation and plant breeding approaches can be used togenerate any plant, expressing any number of components. Presentlypreferred are plants which express collagen alpha 1 and 2 chains, P4H,LH3 and at least one protease (e.g. protease C and/or N). As is furtherdescribed in the Examples section which follows, such plants accumulatecollagen which exhibits stability at temperatures of up to 42° C.

Progeny resulting from breeding or alternatively multiple-transformedplants can be selected, by verifying presence of exogenous mRNA and/orpolypeptides by using nucleic acid or protein probes (e.g. antibodies).The latter approach is preferred since it enables localization of theexpressed polypeptide components (by for example, probing fractionatedplants extracts) and thus also verifies a potential for correctprocessing and assembly. Examples of suitable probes are provided in theExamples section which follows

Once collagen-expressing progeny is identified, such plants are furthercultivated under conditions which maximize expression of the collagenchains as well as the modifying enzymes.

Since free proline accumulation may facilitate over production ofdifferent proline-rich proteins including the collagen chains expressedby the genetically modified plants of the present invention, preferredcultivating conditions are those which increase free prolineaccumulation in the cultivated plant.

Free proline accumulates in a variety of plants in response to a widerange of environmental stresses including water deprivation,salinization, low temperature, high temperature, pathogen infection,heavy metal toxicity, anaerobiosis, nutrient deficiency, atmosphericpollution and UV—irradiation (Hare and Cress, 1997).

Free proline may also accumulate in response to treatment of the plantor soil with compounds such as ABA or stress inducing compounds such ascopper salt, paraquate, salicylic acid and the like.

Thus, collagen-expressing progeny can be grown under different stressconditions (e.g. different concentrations of NaCl ranging from 50 mM upto 250 mM). In order to further enhance collagen production, the effectof various stress conditions on collagen expression will examined andoptimized with respect to plant viability, biomass and collagenaccumulation.

Plant tissues/cells are preferably harvested at maturity, and thecollagen fibers are isolated using well know prior art extractionapproaches, one such approach is detailed below.

Leaves of transgenic plants are ground to a powder under liquid nitrogenand the homogenate is extracted in 0.5 M acetic acid containing 0.2 MNaCl for 60 h at 4° C. Insoluble material is removed by centrifugation.The supernatant containing the recombinant collagen is salt-fractionatedat 0.4 M and 0.7 M NaCl. The 0.7 M NaCl precipitate, containing therecombinant heterotrimeric collagen, is dissolved in and dialyzedagainst 0.1 M acetic acid and stored at −20° C. (following Ruggiero etal., 2000).

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., Eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

Example 1 Constructs and Transformation Schemes

Constructions of expression cassettes and vectors used in this work areillustrated in FIG. 1 a-d. All of the coding sequences in this work wereoptimized for expression in tobacco and chemically synthesized withdesired flanking regions (SEQ ID NOs: 1, 4, 7, 12, 14, 16, 18, 20, 22).FIG. 1 a—the synthetic genes coding for Col1 and Col2 (SEQ ID's 1, 4)fused either to the vacuolar signal or to the apoplast signal (encodedby SEQ ID NO: 7) or without signals were cloned in expression cassettescomposed of a Chrysanthemum rbcS1 promoter and 5′ UTR (SEQ ID NO: 10)and a Chrysanthemum rbcS1 3′UTR and terminator (SEQ ID NO: 11). Thecomplete expression cassettes were cloned in the multiple cloning siteof the pBINPLUS plant transformation vector (van Engelen et al., 1995,Transgenic Res 4: 288-290). FIG. 1 b—The synthetic genes coding for P4Hbeta-human, P4H alpha-human and P4H-plant (SEQ ID NOs: 12, 14 and 16)fused either to the vacuolar signal or to the apoplast signal (encodedby SEQ ID NO: 7) or without signals were cloned in expression cassettescomposed of the CaMV 35S promoter and TMV omega sequence andAgrobacterium Nopaline synthetase (NOS) terminator carried by the vectorpJD330 (Galili et al., 1987, Nucleic Acids Res 15: 3257-3273). Thecomplete expression cassettes were cloned in the multiple cloning siteof the pBINPLUS vectors carrying the expression cassettes of Col1 orCol2. FIG. 1 c—The synthetic genes coding for Proteinase C andProteinase N (SEQ ID NOs: 18, 20) fused either to the vacuolar signal orto the apoplast signal (encoded by SEQ ID NO: 7) were cloned inexpression cassettes composed of a Chrysanthemum rbcS1 promoter and 5′UTR (SEQ ID NO: 10) and a Chrysanthemum rbcS1 3′UTR and terminator (SEQID NO: 11). The complete expression cassettes were cloned in themultiple cloning site of the pBINPLUS plant transformation vector. FIG.1 d—The synthetic gene coding for LH3 (SEQ ID NO: 22) with flankingStrawberry vein banding virus (SVBV) promoter (NCBI accession AF331666REGION: 623 . . . 950 version AF331666.1 GI:13345788) and terminated byAgrobacterium octopin synthase (OCS) terminator (NCBI accession Z37515REGION: 1344 . . . 1538 version Z37515.1 GI:886843) fused either to thevacuolar signal or to the apoplast signal (encoded by SEQ ID NO: 7) orwithout signals was cloned in the multiple cloning site of the pBINPLUSvector carrying the expression cassettes of Col1 and P4H beta.

Co-transformations schemes utilizing the expression cassettes describedin FIG. 1 into a host plant are illustrated in FIG. 2. Each expressioncassette insert is represented by a short name of the coding sequence.The coding sequences and related SEQ ID NOs. are described in Table 1.Each co-transformation is preformed by two pBINPLUS binary vectors. Eachrectangle represents a single pBINPLUS vector carrying one, two or threeexpression cassettes. Promoters and terminators are specified in FIG. 1.

Example 2 Plant Collagen Expression

Synthetic polynucleotide sequences encoding the proteins listed in Table1 below were designed and optimized for expression in tobacco plants.

TABLE 1 List of expressed proteins Included Encoded SwissProt AminoSplicing in SEQ by SEQ Name: accession acids isoform Deletions name IDNO. ID NO. Collagen p02452 1442 One ER signal Col1 3 1 alpha 1(I)version chain [Precursor] Collagen p08123 1342 One ER signal Col2 6 4alpha 2(I) Two changes version chain done in [Precursor] p08123: D549Aand N249I Prolyl 4- p07237 487 One ER signal, P4H 13 12 hydroxylaseversion KDEL betaHuman beta subunit Prolyl 4- p13674 517 P13674-1 ERsignal P4H 15 14 hydroxylase alphaHuman alpha-1 subunit Prolyl 4- Noentry in 252 One Mitochon- P4Hplant 17 16 hydroxylase Swissprot. versiondrial signal Plant NCBI predicted accession: as: aa1-39 gi:15227885Procollagen p13497 866 P13497-1 ER signal, Proteinase 19 18 C-proteinaseBMP1-3 propeptide C Procollagen I o95450 958 O95450-1 ER signal,Proteinase 21 20 N-proteinase LpNPI propeptide N Lysyl o60568 714 One ERsignal LH3 23 22 hydroxylase 3 version

Signal Peptides

(i) Vacuole signal sequence of barley gene for Thiol protease aleurainprecursor (NCBI accession P05167 GI:113603)MAHARVLLLALAVLATAAVAVASSSSFADSNPIRPVTDRAASTLA (SEQ ID NO: 24).

(ii) Apoplast signal of Arabidopsis thaliana endo-1,4-beta-glucanase(Cell, NCBI accession CAA67156.1 GI:2440033); SEQ ID NO. 9, encoded bySEQ ID NO. 7.

Construction of Plasmids

Plant expression vectors were constructed as taught in Example 1, thecomposition of each constructed expression vector was confirmed viarestriction analysis and sequencing.

Expression vectors including the following expression cassettes wereconstructed:

-   1. Collagen alpha 1-   2. Collagen alpha 1+human P4H beta subunit-   3. Collagen alpha 1+human P4H beta subunit+human LH3-   4. Collagen alpha 2-   5. Collagen alpha 2+with human P4H alpha subunit-   6. Collagen alpha 2+with Arabidopsis P4H-   7. Human P4H beta subunit+human LH3-   8. Human P4H alpha subunit    Each of the above described coding sequences was either    translationally fused to a vacuole transit peptide or to an apoplasm    transit peptide or was devoid of any transit peptide sequences, in    which case cytoplasmic accumulation is expected.

Plant Transformation and PCR Screening

Tobacco plants (Nicotiana tabacum, Samsun NN) were transformed with theabove described expression vectors according to the transformationscheme taught in FIG. 2.

Resultant transgenic plants were screened via multiplex PCR using fourprimers which were designed capable of amplifying a 324 bp fragment ofCollagen alpha 1 and a 537 bp fragment of Collagen alpha 2 (Table 2).FIG. 3 illustrates the results of one mulitplex PCR screen.

TABLE 2  List of primers for multiplex PCR foramplification of a 324 bp fragment of Collagenalpha 1 and a 537 bp fragment of Collagen alpha 2 Col1 forward 5′ATCACCAGGAGAACAGGGACCATC 3′ SEQ ID 25 primer (24-mer): Col1 reverse 5′TCCACTTCCAAATCTCTATCCCTAACAAC 3′ SEQ ID 26 primer (29-mer): Col2 forward5′ AGGCATTAGAGGCGATAAGGGAG 3′ SEQ ID 27 primer (23-mer): Col2 reverse 5′TCAATCCAATAATAGCCACTTGACCAC 3′ SEQ ID 28 primer (27-mer):

Example 3 Detection of Human Collagen in Transgenic Tobacco Plants

Total soluble proteins were extracted from tobacco transformants 2, 3and 4 by grinding 500 mg of leaves in 0.5 ml 50 mM Tris-HCl pH=7.5 witha “Complete” protease inhibitor cocktail (product #1836145 from RocheDiagnostics GmbH, 1 tablet per 50 ml buffer). The crude extract wasmixed with 250 μl 4× Sample application buffer containing 10%beta-mercapto-ethanol and 8% SDS, the samples were boiled for 7 minutesand centrifuged for 8 minutes in 13000 rpm. 20 μl of the supernatantwere loaded in a 10% polyacrylamide gel and tested with anti-Collagen I(denatured) antibody ((#AB745 from Chemicon Inc.) in a standard Westernblot procedure (FIG. 4). W.T. is a wild type tobacco. Positive collagenbands are visible in plants that are PCR positive for collagen typeIalpha 1 or alpha 2 or both. Positive control band of 500 ng collagentype I from human placenta (#CC050 from Chemicon Inc.) represents about0.3% of the total soluble proteins (about 150 μg) in the samples fromthe transgenic plants.

Plants expressing collagen at the expected molecular weight up to ˜1% ofthe total soluble proteins were detected when collagen was targeted tothe vacuole (FIG. 4). Subcellular targeting of full length collagen tothe apoplast was sucsessfuly achieved (FIG. 5). Plants expessingcollagen in the cytoplasm (i.e. no targeting peptide) did not accumulatecollagen to detectable levels showing that subcellular tareting ofcollagen in plants is critical for success.

In addition in contrast to the studies of Ruggiero et al. 2000 and Merleet al. 2002 which showed that collagen lacking the N-propeptide wassubjected to significant proteolysis, using the present approach fulllength collagen proteins with C-propeptide and N-propeptide accumulatedin subcellular compartments at high levels.

The present data also clearly shows that crossing two plants eachexpressing a different collagen chain type is advantageous in that itenables selection of plants expressing optimal levels of each chain typeand subsequent plant crossing to achieve the desired collagen producingplant.

Collagen produced by the plants of the present invention includes thenative propeptides and therefore is expected to form a larger proteinthen the human control that was purified by proteolysis. The calculatedmolecular weight of Collagen alpha 1 and alpha 2 chains withouthydroxylations or glycosylations are the following: Col1 withpropeptides −136 kDa, Col1 without propeptides −95 kDa, Col2 withpropeptides −127kDa, Col2 without propeptides −92 kDa.

As can be seen in FIGS. 4, the Col1 bands in transformants 3-5 and 3-49appears larger then Col1 bands in other plants. This indicates prolineshydroxylation in collagen chains by human proline-4-hydroxylaseholoenzyme composed of alpha and beta subunits that were coexpressed inthese plants and targeted to the same subcellular compartment as thehuman collagen chains (e.g. vacuole).

Example 4 Collagen Triple Helix Assembly and Thermal Stability inTransgenic Plants

Assembly of collagen triple helix and the helix thermal stability intransgenic plants were tested by thermal denaturation followed bytrypsin or pepsin digestion of the total crude protein extract oftransgenic plants (FIGS. 6 a-b).

In a first experiment, total soluble proteins from tobacco 2-9(expressing only col alfa1 and no P4H) and 3-5 (expressing both colalfa1+2 and P4H) were extracted by grinding 500 mg leaves in 0.5 ml of50 mM Tris-HCl pH=7.5, centrifuging for 10 minutes in 13000 rpm andcollecting the supernatant. 50 μl of the supernatant were subjected toheat treatment (15 minutes in 33° C. or 43° C.) and then immediatelyplaced on ice. Trypsin digestion was initiated by adding to each sample6 μl of 1 mg/ml Trypsin in 50 mM Tris-HCl pH=7.5. The samples wereincubated for 20 minutes at room temperature (about 22° C.). Thedigestion was terminated by addition of 20 μl 4× sample applicationbuffer containing 10% betamercaptoethanol and 8% SDS, the samples wereboiled for 7 minutes and centrifuged for 7 minutes at 13000 rpm. 500 ofthe supernatant were loaded onto a 10% polyacrylamide gel and testedwith anti-Collagen I antibody ((#AB745 from Chemicon Inc.) using astandard Western blot procedure. Positive controls were samples of ˜500ng human collagen I (#CC050 from Chemicon Inc., extracted from humanplacenta by pepsin digestion) which was added to 50 μl total solubleproteins extracted from w.t. tobacco.

As shown in FIG. 6 a, collagen triple helix that formed in plants #3-5as well as control human collagen was resistant to denaturation at 33°C. In contrast, collagen formed by plants #2-9 denatured at 33° C. Thisdifference in thermal stability indicates a successful triple helixassembly and post translational proline hydroxylation in transformants#3-5 which express both collagen alpha 1 and collagen alpha 2 as well asP4H beta and alpha subunits.

Two bands in transformants #2-9 may represent dimers or trimers, whichare stable following 7 minutes of boiling with SDS and mercaptoethanol.Similar bands are visible in human collagen (upper panel) and intransformants #3-5. A possible explanation is a covalent bond betweentwo peptides in different triple helixes (cross link), formed followingoxidative deamination of two lysines by Lysine oxidase. In a secondexperiment, total soluble proteins from transgenic tobacco 13-6(expressing collagen I alpha 1 and alpha 2 chains—pointed by arrows,human P4H alpha and beta subunits and human LH3) were extracted bygrinding 500 mg of leaves in 0.5 ml of 100 mM Tris-HCl pH=7.5 and 300 mMNaCl, centrifuging for 7 minutes at 10000 rpm and collecting thesupernatant. 50 μl of the supernatant was subjected to heat treatment(20 minutes in 33° C., 38° C. or 42° C.) and then immediately placed onice. Pepsin digestion was initiated by adding to each sample 4.5 μl of0.1M HCl and 4 μl of 2.5 mg/ml Pepsin in 10 mM acetic acid. The sampleswere incubated for 30 minutes at room temperature (about 22° C.). Thedigestion was terminated by adding 5 μl of unbuffered 1 M Tris. Eachsample was mixed with 22 μl 4× Sample application buffer containing 10%beta-mercapto-ethanol and 8% SDS, boiled for 7 minutes and centrifugedfor 7 minutes in 13000 rpm. 40 μl of the supernatant were loaded in a10% polyacrylamide gel and tested with anti-Collagen I antibody ((#AB745from Chemicon Inc.) in a standard Western blot procedure. Positivecontrol was sample of ˜50 ng human collagen I (#CC050 from ChemiconInc., extracted from human placenta by pepsin digestion) added to totalsoluble proteins from w.t. tobacco.

As is illustrated in FIG. 6 b, collagen triple helix that formed inplant #13-6 was resistant to denaturation at 42° C. Cleavage of thepropetides is first visible at 33° C. and gradually increases inefficiency when the temperature is raised to 38° C. and again to 42° C.The cleaved collagen triple helix domain shows a similar migration onthe gel to the migration of the pepsin treated human collagen. The humancollagen that was used in this experiment was extracted from humanplacenta by pepsin proteolysis and therefore lacks the propeptides andsome of the telopeptides.

Example 5 Plant P4H Expression

Induction of Native Plant P4H

Tobacco P4H cDNA was cloned and used as a probe to determine conditionsand treatments that would induce endogenous P4H expression. Northernblot analysis (FIG. 7) clearly shows that P4H is expressed at relativelyhigh levels in the shoot apex and at low levels in leaves. P4H level wasinduced significantly in leaves 4 hours following abrasion treatment(“wounded” in the lower panel). Similar results were achieved usingother stress conditions (not shown).

Detection of Human P4H Alpha and Beta Subunits and Collagen Alpha 1 andAlpha 2 Chains in Transgenic Tobacco Plants

Detection of human P4H alpha and beta subunits and collagen type I alpha1 and alpha 2 chains in transgenic tobacco plants was effected usinganti-human P4H alpha subunit antibody (#63-163 from ICN BiomedicalsInc.), anti-human P4H beta subunit antibody (#MAB2701 from ChemiconInc.) and anti-Collagen I antibody (#AB745 from Chemicon Inc.). Theresults of a western blot probed with these antibodies are shown in FIG.8.

Expression of P4H alpha, P4H beta and collagen I alpha 1 and alpha 2bands was confirmed in plant 13-6 (also transformed also with humanLH3). The calculated molecular weights of P4H alpha and beta includingthe vacuolar signal peptide are 65.5 kDa and 53.4 kDa respectively. Thecalculated molecular weights of Collagen alpha 1 and alpha 2 chains withpropeptides, without hydroxylations or glycosylations are 136 kDa and127 kDa respectively.

Example 6 Vacuolar Targeted Collagen is Stably Expressed in Dark-GrownPlants

Collagen expressing plants—The 20-279 parental tobacco plant line wasgenerated by co-transformation with an expression vector expressingP4Hbeta+LH3 and another expression vector expressing P4H alpha. Eachgene is preceded by a vacuolar targeting determinant of aleurain, aplant vacuolar thiol protease,

The 2-300 parental tobacco plant line was generated by co-transformationwith an expression vector expressing col1 and another expression vectorexpressing col2. Each gene is preceded by a vacuolar targetingdeterminant of aleurain, a plant vacuolar thiol protease.

The 13-652 plant was generated by co-transformation of tobacco plantwith an expression vector encoding Col1, P4Hbeta and LH3 and a secondexpression vector encoding Col2 and P4H alpha. Each gene is preceded bya vacuolar targeting determinant of aleurain, a plant vacuolar thiolprotease, Cassete sequences included in the vectors are described inExample 1 above.

Light and darkness trial—Analysis of six 13-6/52 homozygote plants.Samples from leaf #4+5/6 were taken daily at the same time (12:30) for 8days, from 3 plants that were grown at regular conditions (16 hoursunder light conditions and 8 hours in the dark) and from 3 plants thatwere grown only in the dark.

Total protein extraction and Western blot analysis—Ninety mg of tobaccoleaves were homogenized by mixer mill Type MM301 (Retsch) in anextraction buffer (100 mM Tris HCl pH=7.5, protease inhibitor cocktailavailable from Roche Catalog Number, 04-693-116-001) at 4° C. Following30 min of centrifugation (20,000×g at 4° C.), the supernatant wascollected. Protein samples were fractionated on 8% SDS-PAGE (Laemmli1970) and transferred to a nitrocellulose membrane using BIO-RAD™Protein TRANS-BLOT™ apparatus. The membrane was blocked for 30 min atroom temperature in 3% (g/v) skim milk (Difco), and then reacted witheither commercial rabbit anti-human collagen type I polyclonalantibodies (Chemicon), for over night (o.n.) at room temperature. Themembrane was rinsed with water 3-5 times and then washed for 30 min inTBS. Following incubation with a secondary antibody [goat antirabbit-IgG antibody conjugated to alkaline phosphatase (chemicon)] for 2hours at room temperature, the membrane was rinsed with water for 3-5times and washed for 30 min in TBS. Immunodetection was effected withnitrotetrazolium blue chloride (NBT, Sigma) and5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (BCIP, Sigma), atroom temperature for 2 hour—o.n.

Results

As shown in FIG. 9, tobacco plants transgenic for vacuolar targetedcollagen express Proα1 and Proα2 (lane 1). Collagen from dark grownvacuolar targeted plants exhibited similar stability (lane 2),substantiating the exceptional stability of collagen generated accordingto the teachings of the present invention

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications and GenBank Accession numbers mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application or GenBank

Accession number was specifically and individually indicated to beincorporated herein by reference. In addition, citation oridentification of any reference in this application shall not beconstrued as an admission that such reference is available as prior artto the present invention.

REFERENCES Other References are Cited in the Document

-   1. Bulleid N J, John D C, Kadler K E. Recombinant expression systems    for the production of collagen. Biochem Soc Trans. 2000;28(4):350-3.    Review. PMID: 10961917 [PubMed—indexed for MEDLINE]-   2. Hare P D, Cress W A. Metabolic implications of stress-induced    proline accumulation in plants. Plant Growth Regulation 1997; 21:    79-102.-   3. Hieta R, Myllyharju J. Cloning and characterization of a low    molecular weight prolyl 4-hydroxylase from Arabidopsis thaliana.    Effective hydroxylation of proline-rich, collagen-like, and    hypoxia-inducible transcription factor alpha-like peptides. J Biol    Chem. 2002 Jun. 28; 277(26):23965-71. Epub 2002 Apr. 25. PMID:    11976332 [PubMed—indexed for MEDLINE]-   4. Hulmes D J. Building collagen molecules, fibrils, and    suprafibrillar structures. J Struct Biol. 2002 January-February;    137(1-2):2-10. Review. PMID: 12064927 [PubMed—indexed for MEDLINE]-   5. Inkinen K. Connective tissue formation in wound healing. An    experimental study. Academic Dissertation, September 2003.    University of Helsinki, Faculty of Science, Department of    Biosciences, Division of Biochemistry (ISBN 952-10-1313-3)    http://ethesis.helsinki.fi/julkaisut/mat/bioti/vk/inkinen/-   6. Merle C, Perret S, Lacour T, Jonval V, Hudaverdian S, Garrone R,    Ruggiero F, Theisen M. Hydroxylated human homotrimeric collagen I in    Agrobacterium tumefaciens-mediated transient expression and in    transgenic tobacco plant. FEBS Lett. 2002 Mar. 27; 515(1-3):114-8.    PMID: 11943205 [PubMed—indexed for MEDLINE]-   7. Olsen D, Yang C, Bodo M, Chang R, Leigh S, Baez J, Carmichael D,    Perala M, Hamalainen E R, Jarvinen M, Polarek J. Recombinant    collagen and gelatin for drug delivery. Adv Drug Deliv Rev. 2003    Nov. 28; 55(12):1547-67. PMID: 14623401 [PubMed—in process]-   8. Ruggiero F, Exposito J Y, Bournat P, Gruber V, Perret S, Comte J,    Olagnier B, Garrone R, Theisen M. Triple helix assembly and    processing of human collagen produced in transgenic tobacco plants.    FEBS Lett. 2000 Mar. 3; 469(1):132-6. PMID: 10708770 [PubMed—indexed    for MEDLINE]-   9. Tanaka M, Sato K, Uchida T. Plant prolyl hydroxylase recognizes    poly(L-proline) II helix. J Biol Chem. 1981 Nov. 25;    256(22):11397-400. PMID: 6271746 [PubMed—indexed for MEDLINE]-   10. Wang C, Luosujarvi H, Heikkinen J, Risteli M, Uitto L,    Myllyla R. The third activity for lysyl hydroxylase 3:    galactosylation of hydroxylysyl residues in collagens in vitro.    Matrix Biol. 2002 November; 21(7):559-66. PMID: 12475640    [PubMed—indexed for MEDLINE]

1. A method of producing collagen in a plant or an isolated plant cellcomprising targeting to a vacuole of the plant or the isolated plantcell the collagen alpha 1 chain as set forth in SEQ ID NO: 3 and anexogenous prolyl-4-hydroxylase (P4H) so as to allow hydroxylation of thecollagen alpha 1 chain by said exogenous P4H and not by an endogenousP4H of the plant or isolated plant cell, thereby producing the collagenin the plant.
 2. The method of claim 1, further comprising expressing anexogenous polypeptide selected from the group consisting of Lysylhydroxylase (LH), protease N and protease C.
 3. The method of claim 1,wherein said exogenous P4H comprises a mammalian P4H.
 4. The method ofclaim 1, wherein the plant is selected from the group consisting ofTobacco, Maize, Alfalfa, Rice, Potato, Soybean, Tomato, Wheat, Barley,Canola, Carrot and Cotton.
 5. The method of claim 1, wherein saidexogenous P4H is capable of specifically hydroxylating the Y position ofGly-X-Y triplets of said at least one type of said collagen chain. 6.The method of claim 3, wherein said mammalian P4H comprises a human P4H.7. The method of claim 1, wherein the plant is subjected to a stresscondition.
 8. The method of claim 7, wherein said stress condition isselected from the group consisting of drought, salinity, injury, coldand spraying with stress inducing compounds.
 9. The method of claim 1,further comprising targeting to a vacuole of the plant or the isolatedplant cell the collagen alpha 2 chain as set forth in SEQ ID NO: 6 so asto allow hydroxylation of the collagen alpha 2 chain by said exogenousP4H and not by an endogenous P4H of the plant or isolated plant cell.10. A genetically modified plant or isolated plant cell comprising in avacuole thereof: (i) at least one type of a collagen chain; and (ii) anexogenous P4H.
 11. A method of producing collagen or comprising: (a)providing a plant system comprising: a first genetically modified plantcomprising in a vacuole thereof: (i) the collagen alpha 1 chain as setforth in SEQ ID NO: 3; and (ii) an exogenous P4H; and a secondgenetically modified plant comprising in a vacuole thereof: (i) thecollagen alpha 2 chain as set forth in SEQ ID NO: 6; and (ii) anexogenous P4H; (b) crossing said first plant and said second plant; and(c) selecting progeny expressing said collagen alpha 1 chain and saidcollagen alpha 2 chain thereby producing collagen.
 12. The method ofclaim 11, wherein said exogenous P4H is capable of specificallyhydroxylating the Y position of Gly-X-Y triplets of said collagen alpha1 chain or collagen alpha 2 chain.
 13. The method of claim 11, whereinsaid exogenous P4H is human P4H.
 14. A method of producing collagencomprising: (a) providing the plant system comprising: a firstgenetically modified plant comprising in a vacuole thereof: (i) thecollagen alpha 1 chain as set forth in SEQ ID NO: 3; and (ii) thecollagen alpha 2 chain as set forth in SEQ ID NO: 6; and a secondgenetically modified plant comprising in a vacuole thereof an exogenousP4H; and (b) crossing said first plant and said second plant andselecting progeny expressing the collagen alpha 1 chain, the collagenalpha 2 chain and said P4H thereby producing collagen.